Method for cloning genes

• US 4,394,443 A
• Filed: 12/18/1980
• Issued: 07/19/1983
• Est. Priority Date: 12/18/1980
• Status: Expired due to Fees

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Weissman, Sherman M., Pereira, Dennis, Sood, Ashwani
• Primary Examiner(s): Tanenholtz, Alvin E.
• Assistant Examiner(s): Martinell, James
• Application Number:
  US06/217,643
• Time in Patent Office: 943 Days
• Field of Search: 435/68, 435/172, 435/6, 435/70, 435/91, 435/317, 23/230 B, 536/27, 436/63, 436/501, 436/508
• US Class Current: 435/5
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

A method is provided for isolating and identifying a recombinant clone having a DNA segment therein coding for at least one desired heterologous polypeptide, at least a short amino acid sequence of which is known, by effecting cDNA synthesis on a mixture of mRNAs containing the mRNA coding for the desired polypeptide, isolating the resultant cDNA mixture, inserting the resultant cDNA into recombinant cloning vehicles, transforming hosts with the vehicles, separating the transformants and isolating and identifying a recombinant clone containing a DNA segment which is homologous over at least a portion thereof to at least one oligonucleotide probe specific for the DNA segment; wherein the probe is an extension of the nucleotide sequence of an oligonucleotide primer having a nucleotide sequence complementary to a region of the target mRNA coding for a portion of the known amino acid sequence, and is complementary to a longer region of the target mRNA coding for a longer portion of the known amino acid sequence. Recombinant clones coding for human histocompatibility antigens are provided, in particular, clones for HLA-B antigens.

Claims

1. A method for isolating and indentifying a recombinant clone having a DNA segment therein coding for at least one desired heterologous polypeptide, at least a short amino acid sequence of which is known, said method comprising the steps of:

(a) effecting cDNA synthesis on a mixture of mRNAs containing a target mRNA coding for said at least one polypeptide, and isolating the resultant cDNA mixture;

(b) inserting said resultant cDNA into recombinant cloning vehicles, and transforming hosts with said vehicles; and

(c) separating the transformants and isolating and identifying a recombinant clone containing a DNA segment which is homologous over at least a portion thereof to at least one oligonucleotide probe specific for said DNA segment, said identification being effected by hydridization with said probe;

wherein said probe is an extension of the nucleotide sequence of an oligonucleotide primer having a nucleotide sequence complementary to a region of said target mRNA coding for a portion of said known amino acid sequence, said oligonucleotide probe being produced by separately effecting cDNA synthesis in the presence of said oligonucleotide primer and only three deoxynucleotide triphosphates, isolating and separating extended cDNA fractions having at least three more nucleotides than said primer, sequencing said extended fractions, and recovering at least one oligonucleotide complementary to a longer region of said target mRNA coding for a longer portion of said known amino acid sequence.

2. The method of claim 1, wherein said primer has from 10 to 14 nucleotides.

3. The method of claim 1, wherein said separately effected cDNA synthesis is effected in the further presence of one dideoxynucleoside triphosphate having a different base from said three deoxynucleoside triphosphates;

and wherein said extended cDNA fractions have at least four nucleotides more than said primer.

4. The method of claim 3, wherein said primer has from 10 to 14 nucleotides.

5. The method of claim 1, wherein said target mRNA represents less than 2 mole percent of the total mRNA in the mixture.

6. The method of claim 1, wherein said target mRNA represents up to about 1 mole percent of the total mRNA in the mixture.

7. The method of claim 3, wherein said target mRNA represents up to about 0.1 mole percent of the total mRNA in the mixture.

8. The method of claim 1, wherein said polypeptide is a human major histocompatibility antigen.

9. The method of claim 8, wherein said antigen is coded for on the HLA-B locus.

10. The method of claim 9, wherein said oligonucleotide primer is (5'"'"'^-32 P)dCTTCTCCACAT_OH.

11. The method of claim 3, wherein said polypeptide is a human major histocompatibility antigen coded for on the HLA-B locus;

wherein said oligonucleotide primer is (5'"'"'^-32 P)dCTTCTCCACAT_OH ; and

wherein said probe is (5'"'"'^-32 P)dCTTCTCCACATCACAGCAGCGACCACAGCT_OH.

12. The method of claim 1, which further comprises repeating step (c) at least once, using the oligonucleotide probe from the preceding iteration as a new primer, and recovering an extended oligonucleotide probe complementary to a longer region of said target mRNA coding for a longer portion of said known amino acid sequence.